The discovery of cardiac progenitor cells (CPCs) provides a potential new approach to the treatment of heart failure (HF), The initial results of our first-in-humans clinical study of CPCs in patients with HF (SCIPIO) are encouraging. However, CPC-based therapies are severely limited by the fact that almost all (at least 97%) of the transplanted CPCs die shortly after transplantafion. This implies that increasing the survival of transplanted cells by prevenfing apoptosis will enhance the efficacy of CPC therapy. In the current funding period of this Program Project, we have found that carbon monoxide (CO) and nitric oxide (NO) exert powerful anti-apoptotic actions and form a closely inter-related functional module (CO-NO module), which is regulated by heme oxygenase-1 (HO-1), extracellular superoxide dismutase (ecSOD), and inducible NO synthase (iNOS). We will exploit this discovery to enhance cell-based therapies. The overall goal of Proiect 1 is to elucidate the role of the CO-NO module in regulating CPC function and to evaluate its therapeutic utility after myocardial infarction (Ml). We propose that augmenting this module will greatly potentiate the effectiveness of transplanted CPCs and dramaticallv enhance CPC-mediated cardiac repair. This Project represents the natural evolution of our previous work in this Program Project; having discovered that the CO- NO module affors powerful protection against mmyocardial ischemia, we will now build on this work to enhance the reparative ability of CPCs. In Aim 1, we will determine the roles of HO-1 and CO in modulating CPC function. In Aim 2, we will establish the role of ecSOD in regulating CPC function and mediating HO-1- induced protection of CPCs. In Aim 3, we will determine the role of NO in modulating CPC function and the role of HO-1 and ecSOD in mediating the effects of NO on CPCs. Using both gain- and loss-of-function approaches, in all three Aims we will systematically evaluate fundamental parameters of CPC competence in vitro and the ability of CPCs to repair cardiac damage in vivo in a murine model of post-MI LV remodeling and dysfunction. In Aim 4. we will elucidate the molecular mechanisms whereby CO and NO upregulate ecSOD, and NO upregulates HO-1 and ecSOD in CPCs, focusing on the transcription factor Nrf2. These will be the first studies to examine the role of CO and NO, and their supporting proteins HO-1, ecSOD, and INOS, in modulating CPC function. The results will be entirely new and will add a new dimension to our understanding of CPC biology. In addition, these studies will provide proof-of-principle for the therapeutic utility of manipulations that potentiate the CO-NO module in CPCs, which may lay the groundwork for future trials of genetically or pharmacologically enhanced CPCs in patients with HF. RELEVANCE (See instructions): In 2009 we started SCIPIO, a study of c-kit+ cardiac progenitor cells (CPCs) in patients with heart failure, and the initial results are encouraging. Project 1 builds on SCIPIO. We will test a new therapy - genetically- modified or preconditioned CPCs, which promise to be superior to the cells currently being used in SCIPIO. If the results confirm our working hypothesis, these studies will lay the groundwork for testing more effective CPCs for the treatment of heart failure and thus may facilitate translation of cell therapies to humans.